The present invention relates to a promoter for a xcex941-pyrroline-5-carboxylate synthetase gene in Arabidopsis thaliana (referred as P5CS hereafter). Gene recombination is useful for the development of new plants or for the reproduction of culture cell so as to produce useful material.
It is known that many plants, including halophytes, accumulate compatible proline when they are exposed to osmotic stress. The fact suggests that the proline accumulated in plant organs plays a role in keeping water in the plant organs.
In plants, proline is produced from glutamic acid via both enzymes of P5CS and xcex941-pyrroline-5-carboxylate reductase (referred as P5CR hereafter). In a plant that has accumulated proline in its organs, when the activity of P5CS and the expression level of the P5CS gene increase as when they are exposed to water stress (dry stress or osmotic stress which means that plant has difficulty getting water), the activity of P5CR and the expression level of the P5CR gene do not change and remain at a low level. This suggests that the expression of P5CS controls a rate-limiting step of the production of the proline in plants under water stress (Yoshiba et al. Plant J. 7: 751-760 (1995)). Further, the expression of P5CS gene is induced not only under water stress such as dry stress or osmotic stress, but also by an abscisic acid (ABA)-dependent pathway. And the expression of the P5CS gene is suppressed in the absence of osmotic stress (Yoshiba et al. Plant Cell Physiol. 38:1095-1102 (1997)).
A conventional promoter based on a vector for plant is a promoter derived from a gene of a cauliflower mosaic virus (CaMV), or a promoter derived from a gene of a Ti plasmid of agrobacterium tumefacience (which is a kind of soil microorganism). These promoters are used as a promoter which effectively expresses a gene introduced into a plant.
The technology for introducing of gene into a plant is generally known. Further, a promoter which effectively expresses a gene introduced into the plant is also developed and used. The promoter based on a vector for the plant is used as an example. FIG. 1A shows that a promoter 101 is combined with a gene introduced into a plant so that a gene expression is induced by the promoter.
However, these conventional promoters express the respective gene after it is introduced into a plant regardless of a growing period or any part of the plant. A plant""s growing usually does not require such a gene expression, and a gene expression is not good, in many cases, for a plant""s growing. So if it is possible to control gene expression according to a desired condition, it is very useful for a plant""s growing. Namely, if it is possible to realize a promoter specified to a growing period, a promoter specified to a tissue of plant or a promoter specified to an environmental circumstance, an expression of a gene combined to the down stream of the promoter is arbitrarily controlled to happen only when it is needed.
FIGS. 1B-FIG. 1E show the above. FIG. 1B shows a status in which gene 102 is combined with a promoter 103 responsive to an environmental circumstance. FIG. 1C shows a status in which gene 104 is combined with a promoter 105 specified to a tissue of plant. FIG. 1D shows a status in which gene 106 is combined with a promoter 107 specified to a growing period. Further, FIG. 1E shows a status in which genes 102, 104 and 106 is combined sequentially then introduced into a plant, and in which the above promoters specified to each condition are sequentially introduced at the upper stream of the combined genes.
According to the above, the present invention provides a promoter of the P5CS gene which controls a gene expression responsive to an environmental circumstance, namely, turning on a gene expression when an environmental circumstance becomes wrong and turning off the gene expression when the environmental circumstance becomes right. The promoter of the P5CS gene of the present invention controls to induce or suppress the expression of a useful gene, such as a gene resisting an environmental stress, connected to down stream of the promoter. Therefore, when there is a change of an earth environment, it is possible to control whether a plant resists against such an environmental stress. Further, if the promoter of the P5CS gene is combined with a gene which dyes a tissue of the plant, it is possible to monitor a status change of the tissue corresponding to environmental circumstance, i.e., a sensor plant corresponding to an environmental stress.
It is known that many plants accumulate proline, a kind of amino acid, in cells when they are subject to water stress, in which the plant has difficulty getting water, such as through drought or salinity stress. The proline is synthesized from two enzymes of P5CS xe2x80x9cxcex941-pyrroline-5-carboxylate synthetasexe2x80x9d xcex941- and P5CR xe2x80x9c-pyrroline-5-carboxylate reductasexe2x80x9d. It is known that P5CS is a rate-limiting step of proline biosynthesis under water stress. Further, it is known that the P5CS gene is rapidly induced to express under water stress, such as dry stress or osmotic stress, and given an abscisic acid (ABA), and the P5CS gene is rapidly suppressed to express in the absence of the stress and the abscisic acid (ABA).
As such, the inventor came up with an idea that the promoter of the P5CS gene may induce an expression of a gene corresponding to environmental circumstance. Namely, the expression of a gene is controlled by the P5CS promoter via giving to a plant or removing from the plant an environmental stress. Cloning and sequence analysis of genomic DNA including the promoter region of the P5CS gene from Arabidopsis thaliana are executed.
The promoter region of the P5CS gene is taken out and combined with a xcex2-glucuronidase gene so that the chimeric gene is introduced into a plant. An expression of the xcex2-glucuronidase gene of a plant introduced in the chimeric gene is induced under dehydration stress or osmotic stress via abscisic acid (ABA) biosynthesis.
The promoter of the P5CS gene can control an expression of a gene under water stress. Namely, the present invention provides a DNA including the promoter of the P5CS gene. Further, the present invention provides a vector for a plant including the above DNA.